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Activity of Rho-family GTPases during cell division as visualized with FRET-based probes.

Yoshizaki H, Ohba Y, Kurokawa K, Itoh RE, Nakamura T, Mochizuki N, Nagashima K, Matsuda M - J. Cell Biol. (2003)

Bottom Line: Cell.Biol. 22:6582-6591).The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Japan.

ABSTRACT
Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)-based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582-6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.

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Localization and FRET imaging of Raichu probes. (A) HeLa cells expressing GFP-tagged Rho-family GTPases of the wild-type (WT) or constitutively active form (Active) are presented in the first two columns. The next two columns show HeLa cells expressing Raichu probes with either the authentic carboxy termini (CT) or K-Ras4B CT. A cell image of Raichu-RBD–expressing cells is also shown at the bottom of A. Cells were excited by an Argon laser at a 488-nm wavelength and imaged with a confocal microscope. (B) Ratio imaging by TPEM. HeLa cells expressing the Raichu probes listed on the left were imaged for YFP (535 ± 12 nm) and CFP (480 ± 15 nm) with a 790-nm excitation wavelength. XY planes from the bottom to the top of each cell were stacked, and the YFP/CFP ratio image is used to show the FRET efficiency in the intensity-modulated display mode. XZ sections were prepared along the dotted white lines. White bars indicate 10 μm (A and B). The upper and lower limits of the ratio range are shown at the right of each panel.
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fig3: Localization and FRET imaging of Raichu probes. (A) HeLa cells expressing GFP-tagged Rho-family GTPases of the wild-type (WT) or constitutively active form (Active) are presented in the first two columns. The next two columns show HeLa cells expressing Raichu probes with either the authentic carboxy termini (CT) or K-Ras4B CT. A cell image of Raichu-RBD–expressing cells is also shown at the bottom of A. Cells were excited by an Argon laser at a 488-nm wavelength and imaged with a confocal microscope. (B) Ratio imaging by TPEM. HeLa cells expressing the Raichu probes listed on the left were imaged for YFP (535 ± 12 nm) and CFP (480 ± 15 nm) with a 790-nm excitation wavelength. XY planes from the bottom to the top of each cell were stacked, and the YFP/CFP ratio image is used to show the FRET efficiency in the intensity-modulated display mode. XZ sections were prepared along the dotted white lines. White bars indicate 10 μm (A and B). The upper and lower limits of the ratio range are shown at the right of each panel.

Mentions: Before FRET imaging, the intracellular localizations of the probes were compared with those of the Rho-family GTPases tagged with GFP (Fig. 3 A). The distribution of GFP-tagged Rho-family GTPases was in general agreement with the previous report (Michaelson et al., 2001); GFP–RhoA–WT was observed mostly in the cytoplasm, and GFP–Rac1–WT and GFP–Cdc42–WT were observed at both the plasma membrane and intracellular membrane compartments. The active forms of GFP-tagged Rho-family GTPases were detected both at the plasma membrane and the intracellular membrane compartments. The distribution of Raichu–RhoA/RhoA–CT, Raichu–Rac1/Rac1–CT, and Raichu–Cdc42/Cdc42–CT was indistinguishable from those of the constitutively active mutants, supporting the previous finding that the carboxy-terminal region primarily determines the localization of the Rho-family GTPases in their active forms (Michaelson et al., 2001; Fig. 3 A, third column). In a clear contrast, all probes with the carboxy terminus of K-Ras4B were localized mainly at the plasma membrane (Fig. 3 A, fourth column). The distribution of Raichu-RBD was typical for a cytosolic protein (Fig. 3 A, bottom).


Activity of Rho-family GTPases during cell division as visualized with FRET-based probes.

Yoshizaki H, Ohba Y, Kurokawa K, Itoh RE, Nakamura T, Mochizuki N, Nagashima K, Matsuda M - J. Cell Biol. (2003)

Localization and FRET imaging of Raichu probes. (A) HeLa cells expressing GFP-tagged Rho-family GTPases of the wild-type (WT) or constitutively active form (Active) are presented in the first two columns. The next two columns show HeLa cells expressing Raichu probes with either the authentic carboxy termini (CT) or K-Ras4B CT. A cell image of Raichu-RBD–expressing cells is also shown at the bottom of A. Cells were excited by an Argon laser at a 488-nm wavelength and imaged with a confocal microscope. (B) Ratio imaging by TPEM. HeLa cells expressing the Raichu probes listed on the left were imaged for YFP (535 ± 12 nm) and CFP (480 ± 15 nm) with a 790-nm excitation wavelength. XY planes from the bottom to the top of each cell were stacked, and the YFP/CFP ratio image is used to show the FRET efficiency in the intensity-modulated display mode. XZ sections were prepared along the dotted white lines. White bars indicate 10 μm (A and B). The upper and lower limits of the ratio range are shown at the right of each panel.
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Related In: Results  -  Collection

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fig3: Localization and FRET imaging of Raichu probes. (A) HeLa cells expressing GFP-tagged Rho-family GTPases of the wild-type (WT) or constitutively active form (Active) are presented in the first two columns. The next two columns show HeLa cells expressing Raichu probes with either the authentic carboxy termini (CT) or K-Ras4B CT. A cell image of Raichu-RBD–expressing cells is also shown at the bottom of A. Cells were excited by an Argon laser at a 488-nm wavelength and imaged with a confocal microscope. (B) Ratio imaging by TPEM. HeLa cells expressing the Raichu probes listed on the left were imaged for YFP (535 ± 12 nm) and CFP (480 ± 15 nm) with a 790-nm excitation wavelength. XY planes from the bottom to the top of each cell were stacked, and the YFP/CFP ratio image is used to show the FRET efficiency in the intensity-modulated display mode. XZ sections were prepared along the dotted white lines. White bars indicate 10 μm (A and B). The upper and lower limits of the ratio range are shown at the right of each panel.
Mentions: Before FRET imaging, the intracellular localizations of the probes were compared with those of the Rho-family GTPases tagged with GFP (Fig. 3 A). The distribution of GFP-tagged Rho-family GTPases was in general agreement with the previous report (Michaelson et al., 2001); GFP–RhoA–WT was observed mostly in the cytoplasm, and GFP–Rac1–WT and GFP–Cdc42–WT were observed at both the plasma membrane and intracellular membrane compartments. The active forms of GFP-tagged Rho-family GTPases were detected both at the plasma membrane and the intracellular membrane compartments. The distribution of Raichu–RhoA/RhoA–CT, Raichu–Rac1/Rac1–CT, and Raichu–Cdc42/Cdc42–CT was indistinguishable from those of the constitutively active mutants, supporting the previous finding that the carboxy-terminal region primarily determines the localization of the Rho-family GTPases in their active forms (Michaelson et al., 2001; Fig. 3 A, third column). In a clear contrast, all probes with the carboxy terminus of K-Ras4B were localized mainly at the plasma membrane (Fig. 3 A, fourth column). The distribution of Raichu-RBD was typical for a cytosolic protein (Fig. 3 A, bottom).

Bottom Line: Cell.Biol. 22:6582-6591).The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Virology, Research Institute for Microbial Diseases, Osaka University, Japan.

ABSTRACT
Rho-family GTPases regulate many cellular functions. To visualize the activity of Rho-family GTPases in living cells, we developed fluorescence resonance energy transfer (FRET)-based probes for Rac1 and Cdc42 previously (Itoh, R.E., K. Kurokawa, Y. Ohba, H. Yoshizaki, N. Mochizuki, and M. Matsuda. 2002. Mol. Cell. Biol. 22:6582-6591). Here, we added two types of probes for RhoA. One is to monitor the activity balance between guanine nucleotide exchange factors and GTPase-activating proteins, and another is to monitor the level of GTP-RhoA. Using these FRET probes, we imaged the activities of Rho-family GTPases during the cell division of HeLa cells. The activities of RhoA, Rac1, and Cdc42 were high at the plasma membrane in interphase, and decreased rapidly on entry into M phase. From after anaphase, the RhoA activity increased at the plasma membrane including cleavage furrow. Rac1 activity was suppressed at the spindle midzone and increased at the plasma membrane of polar sides after telophase. Cdc42 activity was suppressed at the plasma membrane and was high at the intracellular membrane compartments during cytokinesis. In conclusion, we could use the FRET-based probes to visualize the complex spatio-temporal regulation of Rho-family GTPases during cell division.

Show MeSH